Stabilized antibody compositions

ABSTRACT

The invention provides novel compositions of antibodies based on liquid vehicles selected from semifluorinated alkanes. The use of these vehicles provides for improved stability and shelf-life of antibodies and their derivatives. The compositions are useful for topical administration or for parenteral injection.

FIELD

The present invention is in the field of antibody compositions, inparticular compositions which are useful as pharmaceutical formulationsfor therapeutic or diagnostic use.

BACKGROUND

Antibody-based therapies have come to the forefront in recent years aseffective treatment options for numerous diseases such as cancer orautoimmune diseases, in conjunction with many new developments inantibody engineering and production technologies.

One of the major challenges associated with antibody formulation anddelivery is maintaining the stability and form of the antibodytherapeutic, in particular for long-term storage and for transport.Antibodies, like other types of protein-based therapeutics, aresusceptible to physical and chemical instability under stress conditionssuch as temperature changes from freeze-thawing or during transport,exposure to light, oxygen or chemical/solvents, shear stress, and pHstress.

They may undergo denaturation (e.g. loss of tertiary and/or secondarystructure) or interact to form aggregates. While antibody aggregationcan occur in both the liquid and solid state, it is especiallyproblematic in liquid formulations, especially at high concentrations ofantibody. Antibodies are typically therapeutically effective atrelatively high doses. High antibody concentrations, such as to minimizedose volumes and to make administration more patient-friendly (e.g.subcutaneous injection instead of intravenous infusion, decreased numberof injections) are therefore generally desirable in pharmaceuticalformulations.

Aggregation, in effect, can lead to loss of active antibody therapeutic,leading to unreliable and ineffective dosing. More significantly,aggregates may also exhibit toxicity and trigger undesirable or seriousimmunogenic responses. Aggregation resulting in the precipitation oflarge particulates which impede flow is undesirable for any kind ofparenteral application.

Antibody modification and degradation via chemical pathways such asoxidation, deamidation, isomerization, disulfide bond formation andother irreversible crosslinking reactions may also occur over time andlead to inactivation of the antibody, as well as trigger aggregation.These reactions, along with aggregation, are often accelerated atelevated temperatures; refrigeration is consequently almost always aprerequisite. In many of these chemical reactions, water also plays asignificant role either as a mediator or as a reactive intermediate suchas in the hydrolytic cleavage of amide bonds. The exclusion of water,such as by lyophilization, or freeze-drying, to form a solid-statepowder formulation may thus be an effective measure towards preparing astable formulation of an antibody therapeutic.

Some of the formulations of marketed therapeutic antibody or antibodyderivatives/fragments are based on lyophilized powder formulations whichneed to be reconstituted under sterile conditions with aqueous mediashortly prior to administration by a trained medical or paramedicalpractitioner. For example, omalizumab (e.g. Xolair®, marketed byGenentech) is available as a lyophilized powder in a single-use vial.

The reconstitution of the lyophilized antibody in sterile aqueous mediaas an extra step prior to actual administration, however, carries therisk of improper handling (e.g. shaking) or dosing, as well ascontamination. The reconstitution step itself may trigger aggregation ifthe pH or temperature of the aqueous medium is suboptimal, the timeallowed for rehydration is too short or the vial is too aggressivelyshaken during the dissolving step. The propensity for waste is alsohigher, as failure to properly dissolve the lyophilized antibody productwithin the recommended time period usually requires for the sample to bediscarded.

It should be noted that the lyophilization process step itself mayinduce aggregation and/or degradation. Additional stabilizing excipientssuch as saccharides or polyols are often added to the pre-lyophilizationcomposition, along with other excipients such as bulking agents. Theaddition of other excipients may also be required after lyophilizationin order to support the longer shelf-life of the antibody, adding to thenumber of components in the final formulation.

Ready-to-use liquid formulations would generally be preferred by theusers, due to the ease of preparation for administration. If stable, aliquid formulation is also attractive for the pharmaceuticalmanufacturer due to the avoidance of lyophilisation, which istime-consuming and costly both during drug development and routinemanufacture. Indeed, many marketed formulations of antibodies orantibody derivatives are aqueous-based solutions. With aqueousformulations, the pH of the medium can have a significant impact on thestability of the antibody in terms of promoting or reducing thelikelihood of various degradative chemical reactions. Consequently, anoptimized buffering system is always required, along with otherformulation excipients such as antioxidant free-radical scavengers,surfactants and other anti-aggregation additives, or preservatives inorder to provide stabilization to the antibody and counteract thevarious possible degradation processes that may occur during storage inan aqueous environment over time.

Alternative formulation options to lyophilization and aqueous solutionsare also known, such as the use of non-aqueous liquids as carriervehicles. For example, WO2012/121754 describes non-aqueous, highconcentration suspension formulations comprising a hydrophobic vehiclesuch as sesame oil, and a viscosity-reducing agent such as ethyl oleate,and an anti-TNFα antibody. These compositions require addition of aviscosity-reducing agent that is fully miscible in the carrier, in orderto make the oil carriers more amenable towards injection. Generally, theparenteral use of lipids and oils can cause pain and other undesirableside effects at the injection site. These types of compounds may alsoslow down the release of therapeutic agent, and are not ideal if rapidor immediate bioavailable is preferred.

Perfluorinated compounds have also been described as possiblenon-aqueous liquid carriers of biologically active agents such asproteins and peptides. For example, U.S. Pat. No. 6,458,376 describescompositions proposed for ophthalmic applications (such as topicallyapplied eye drops) in which therapeutic/diagnostic compounds, includingoligopeptides and protein growth factors are suspended inperfluorocarbons or fluorinated silicone oils and in the presence of atleast one surfactant. There is no mention, however, of such compositionscomprising antibodies or antibody fragments and derivatives.

U.S. Pat. No. 6,730,328 describes thermally stable formulations in whichnon-aqueous, hydrophobic, non-reactive vehicles such as mineral oil,perfluorodecalin, methoxyflurane, perfluorotributylamine and tetradecaneare used for suspension compositions comprising proteins, proteinaceouscompounds and nucleic acids. The formulations are proposed forparenteral, transdermal, mucosal, oral and enteral methods ofadministration, as well as their use for long-term continuousadministration and delivery via an implantable device. However, nospecific example of a suspension of an antibody or antibody fragment orderivative in such vehicles is disclosed, nor is there any teaching withregard to the stability of such compositions at elevated temperaturesbeyond a three month time point. The actual tissue compatibility ofthese types of compositions has not been demonstrated either.

WO 2011/073134 discloses solutions of ciclosporin, a cyclic polypeptidewith a molecular weight of 1202.31 in a semifluorinated alkane,optionally in the presence of a cosolvent such as ethanol. Whilstsuspensions and emulsions are mentioned as optional alternatives, thereis no specific disclosure of such type of composition, or anycomposition comprising high molecular weight proteinaceous species inthe kiloDalton range such as antibodies or antibody fragments orderivatives.

It is therefore an object of the present invention to introduce novelantibody compositions which overcome any of the limitations anddisadvantages associated with formulations currently known in the art.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides a novel composition of anantigen-binding polypeptide or protein, and a liquid vehicle whichcomprises a semifluorinated alkane of the formula RFRH, wherein RF is alinear perfluorinated hydrocarbon segment with 4 to 12 carbon atoms, andwherein RH is a linear alkyl group with 4 to 8 carbons. Theantigen-binding polypeptide or protein is incorporated in thecomposition such as to form a dispersion or suspension.

In another aspect, the antigen-binding polypeptide or protein may beselected from a monoclonal antibody, an antibody fragment, a polyclonalantibody, a fusion protein comprising an antibody fragment, or anantibody-drug conjugate.

In a further aspect, the invention provides a method for the treatment,prevention or diagnosis of a disease or condition in a patient in needthereof, comprising the step of administering a composition comprisingan antigen-binding polypeptide or protein, and a liquid vehicle whichcomprises a semifluorinated alkane of the formula RFRH, wherein RF is alinear perfluorinated hydrocarbon segment with 4 to 12 carbon atoms, andwherein RH is a linear alkyl group with 4 to 8 carbons, and wherein theantigen-binding polypeptide or protein is incorporated in thecomposition such as to form a dispersion or suspension.

In yet another embodiment, the invention provides for a method ofstabilizing an antigen-binding polypeptide or protein, comprising thestep of mixing the antigen-binding polypeptide or protein with a liquidvehicle comprising a semifluorinated alkane of the formula RFRH, whereinRF is a linear perfluorinated hydrocarbon segment with 4 to 12 carbonatoms, and wherein RH is a linear alkyl group with 4 to 8 carbons, toform a suspension or dispersion.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Binding activities of anti-ECSCR antibody in F6H8 stored at 25°C. or 40° C. for 3 or 6 months compared to initial time point ofreconstitution and to control samples of lyophilized antibody samplesstored at −80° C. and never-lyophilized samples stored in PBS at 4° C.(Example 1).

FIG. 2. Binding activity of bevacizumab antibody stored in F4H5 or F6H8between 50° C. and 80° C., compared to the antibody stored in aqueousPBS buffer (Example 3).

FIG. 3. Binding activity of Fsn1006 antibody stored in F4H5 or F6H8between 50° C. and 80° C., compared to the antibody stored in aqueousPBS buffer (Example 3).

FIG. 4. Binding activity of Fsn0503 antibody stored in F4H5 or F6H8between 50° C. and 80° C., compared to the antibody stored in aqueousPBS buffer (Example 3).

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the present invention provides compositionscomprising an antigen-binding polypeptide or protein and a liquidvehicle which comprises a semifluorinated alkane of the formula RFRH,wherein RF is a linear perfluorinated hydrocarbon segment with 4 to 12carbon atoms, and wherein RH is a linear alkyl group with 4 to 8carbons. Moreover, the antigen-binding polypeptide or protein isincorporated in the composition such as to form a dispersion or asuspension; i.e. the antigen-binding polypeptide or protein is dispersedor suspended in the liquid vehicle.

Semifluorinated alkanes provide a number of advantages from thepharmaceutical perspective. They are substantially non-toxic and arefound to be well-tolerated by various types of human and animal tissuewhen administered topically or parenterally. In addition, they arechemically inert and are generally compatible with active and inactiveingredients in pharmaceutical formulations. They are also capable ofdissolving a large range of compounds, such as small molecule activeingredients and many common pharmaceutically acceptable excipients,probably due to their inherent amphiphilicity. Moreover, semifluorinatedalkanes, when acting as vehicles for compounds that are not soluble orpoorly soluble (such as antigen-binding proteins or polypeptides), formdispersions or suspensions with very useful physical or pharmaceuticalproperties, i.e. with little or no tendency to form solid,non-dispersible sediments.

It has been found by the inventors that the presence of asemifluorinated alkane as a liquid vehicle in a composition comprisingan antigen-binding protein or polypeptide has a remarkable stabilizingeffect on these components. In particular, compositions comprisingsemifluorinated alkane as a liquid vehicle are capable of substantiallypreventing or inhibiting their aggregation and reducing chemicaldegradation over a substantial period of time, at room temperature andeven at higher temperatures such as 40° C., without loss of biologicalactivity.

It has also been found that antigen-binding protein dispersions andsuspensions in semifluorinated alkanes exhibit a remarkable degree ofphysical stability. The occurrence of flotation or sedimentation takesplace slowly, leaving sufficient time for the withdrawal of a dose aftergentle shaking or swirling of the container (e.g. a vial) with thedispersion or suspension. The antigen-binding protein particles insemifluorinated alkane appear to largely retain their original particlesize distribution, and are readily redispersible; poorly re-dispersibleaggregates do not appear to be formed. Importantly, this provides for ahigher level of dosing accuracy in terms of precision andreproducibility.

In contrast, suspensions or dispersions in other chemically inertvehicles tend to be unstable, leading to formation of dense and poorlyredispersible aggregates, and making precise dosing challenging, or insome cases, impossible, such as leading to the clogging of fine-gaugedneedles typically used for subcutaneous injections. Aggregated proteinparticles also present a high risk towards triggering adverseimmunogenic reactions.

Suspensions or dispersions that are unstable tend to separate quickly byflotation of the dispersed phase, or by its sedimentation, depending onthe relative densities of the dispersed phase and of the continuousphase. Such behaviour is usually accompanied by rapid formation of denseand poorly re-dispersible aggregates. Rapid flotation or sedimentationmakes accurate and reproducible dosing challenging, if not impossible.For example, if an injectable or ophthalmic suspension settles rapidlyafter shaking, and if a first dose from the full container is notwithdrawn immediately after shaking, a dose that is withdrawn maycontain a lower-than-intended number of drug particles (or if thecontainer is held upside down, a large-than-intended dose will bedispensed). Later doses withdrawn from the same container will also thencontain either too high or too low of a drug-dose per volume. Vigorousshaking of antigen-binding polypeptides and proteins in attempt tore-disperse poorly re-dispersible aggregates may also further triggertheir further aggregation and deterioration.

Key advantages of the present invention are brought about by thepresence of a semifluorinated alkane in the composition, functioning asa liquid vehicle. The advantageous properties of semifluorinatedalkane-based suspensions result in superior pharmaceutical quality andperformance characteristics, and also increase the convenience of usefor the patient and/or the healthcare provider.

Semifluorinated alkanes are linear or branched alkanes some of whosehydrogen atoms have been replaced by fluorine. In the semifluorinatedalkanes (SFAs) used in the present invention, one linear non-fluorinatedhydrocarbon segment and one linear perfluorinated hydrocarbon segmentare present. These compounds thus follow the general formulaF(CF₂)_(n)(CH₂)_(m)H. According to the present invention, n is selectedfrom the range of 4 to 12, and m is selected from the range of 4 to 8.

A nomenclature which is frequently used for semifluorinated alkanesdesignates a perfluorated hydrocarbon segment as RF and anon-fluorinated segment as RH. Alternatively, the compounds may bereferred to as FnHm and FnHm, respectively, wherein F means aperfluorated hydrocarbon segment, H means a non-fluorinated segment, andn and m define the number of carbon atoms of the respective segment. Forexample, F3H3 is used for perfluoropropylpropane, F(CF₂)₃(CH₂)₃H.Moreover, this type of nomenclature is usually used for compounds havinglinear segments. Therefore, unless otherwise indicated, it should beassumed that F3H3 means 1-perfluoropropylpropane, rather than2-perfluoropropylpropane, 1-perfluoroisopropylpropane or2-perfluoroisopropylpropane.

Preferred semifluorinated alkanes include in particular the compoundsF4H5, F4H6, F4H8, F6H4, F6H6, F6H8, and F6H10. Particularly preferredfor carrying out the invention are F4H5, F4H6, F6H6 and F6H8. In anotherparticularly preferred embodiment, the composition of the inventioncomprises F6H8.

Optionally, the composition may comprise more than one SFA. It may beuseful to combine SFAs, for example, in order to achieve a particulartarget property such as a certain density or viscosity. If a mixture ofSFAs is used, it is furthermore preferred that the mixture comprises atleast one of F4H5, F4H6, F6H4, F6H6, F6H8, and F6H10, and in particularone of F4H5, F4H6, F6H6 and F6H8. In another embodiment, the mixturecomprises at least two members selected from F4H5, F4H6, F6H4, F6H6,F6H8, and F6H10, and in particular at least two members selected fromF4H5, F6H6 and F6H8.

Liquid SFAs are chemically and physiologically inert, colourless andstable. Their typical densities range from 1.1 to 1.7 g/cm³, and theirsurface tension may be as low as 19 mN/m. SFAs of the RFRH type areinsoluble in water but also somewhat amphiphilic, with increasinglipophilicity correlating with an increasing size of the non-fluorinatedsegment.

Liquid SFAs of the RFRH type are being used commercially for unfoldingand reapplying a retina, for long-term tamponade as vitreous humoursubstitute (H. Meinert et al., European Journal of Ophthalmology, Vol.10(3), pp. 189-197, 2000), and as wash-out solutions for residualsilicon oil after vitreo-retinal surgery. Experimentally, they have alsobeen used as blood substitutes (H. Meinert et al., Biomaterials,Artificial Cells, and Immobilization Biotechnology, Vol. 21(5), pp.583-95, 1993). These applications have established SFAs asphysiologically well tolerated compounds. On the other hand, SFAs havenot been used as excipients in approved drug products as of today.

The composition of the invention comprises an antigen-bindingpolypeptide or protein. Polypeptides and proteins in general representpolymers of amino acid units that linked to each other by peptide bonds.Since the size boundaries that are often used to differentiate betweenpolypeptides and proteins are somewhat arbitrary, the two expressionsfor these molecules should—within the context of the presentinvention—not be understood as mutually exclusive: A polypeptide mayalso be referred to as a protein, and vice versa. Typically, the term“polypeptide” only refers to a single polymer chain, whereas theexpression “protein” may also refer to two or more polypeptide chainsthat are linked to each other by non-covalent bonds.

More specifically, and as used within the context of the presentinvention, antigen-binding polypeptides or proteins refer to full-lengthand whole antibodies (also known as immunoglobulins) in their monomer,or polymeric forms and any fragments, chains, domains or anymodifications derived from a full-length antibody capable ofspecifically binding to an antigen. The antigen-binding polypeptides orproteins may belong to any of the IgG, IgA, IgD, IgE, or IgMimmunoglobulin isotypes or classes. Fusion proteins comprising anantibody fragment capable of specifically binding to an antigen andantibody-drug conjugates are also within the definition ofantigen-binding polypeptides or proteins as used herein.

A full-length antibody is a Y-shaped glycoprotein comprising of ageneral structure with an Fc (fragment crystallisable) domain and a Fab(fragment antigen binding) domain. These are structurally composed fromtwo heavy (H) chains and two light (L) chain polypeptide structuresinterlinked via disulfide bonds to form the Y-shaped structure. Eachtype of chain comprises a variable region (V) and a constant region (C);the heavy chain comprises a variable chain region (V_(H)) and variousconstant regions (e.g. C_(H)1, C_(H)2, etc.) and the light chaincomprises a variable chain region (V_(L)) and a constant region (CO. TheV regions may be further characterized into further sub-domains/regions,i.e. framework (FR) regions comprising more conserved amino acidresidues and the hypervariable (HV) or complementarity determiningregions (CDR) which comprise of regions of increased variability interms of amino acid residues. The variable regions of the chainsdetermine the binding specificity of the antibody and form theantigen-binding Fab domains of an antibody.

In a preferred embodiment of the invention, the compositions comprise anantigen-binding polypeptide or protein, wherein the antigen-bindingpolypeptide or protein is selected from a monoclonal antibody,polyclonal antibody, an antibody fragment, a fusion protein comprisingan antibody fragment, an antibody-drug conjugate, or any combinationthereof.

In a particularly preferred embodiment of the invention, thecompositions comprise an antigen-binding polypeptide or protein selectedfrom a monoclonal antibody (mAb). A monoclonal antibody refers to anantibody obtained from a homogenous population of antibodies that arespecific towards a single epitope or binding site on an antigen.Monoclonal antibodies may be produced using antibody engineeringtechniques known in the art, such as via hybridoma or recombinant DNAmethods.

Also within the scope of antigen-binding polypeptides and proteins, andmonoclonal antibodies are antibody fragments. As defined herein,antibody fragments include any region, chain, domain of an antibody, orany constructs or conjugates thereof that can interact and bindspecifically to an antigen, and may be monovalent, bivalent, or evenmultivalent with respect to binding capability. Such antibody fragmentsmay be produced from methods known in the art, for example, dissection(e.g. by proteolysis) of a full-length native antibody, from proteinsynthesis, genetic engineering/DNA recombinant processes, chemicalcross-linking or any combinations thereof. Antibody fragments arecommonly derived from the combination of various domains or regionsfeatured in variable V region of a full-length antibody.

In an embodiment of the invention, the compositions comprise anantigen-binding polypeptide or protein selected from an antibodyfragment, wherein the antibody fragment is a fragment antigen-binding(Fab), a single-chain variable fragment (scFv), a single-domainantibody, a minibody, or a diabody.

Particularly preferred antibody fragments are fragment antigen-bindingdomains (Fab, also referred to as Fab′) or Fab dimers comprising of twoFab fragments linked by a disulfide linkage. Examples of Fabs areabciximab, certolizumab, digifab, and ranibizumab. A preferred Fab iscertolizumab (also known as certolizumab pegol), which is a recombinanthumanized antibody Fab′ fragment conjugated to polyethylene glycol.Certolizumab has a molecular mass of 91 kDa and is directed againsttumour necrosis factor alpha (TNFα).

In yet another embodiment of the invention, the compositions maycomprise a single-chain variable fragment (scFv) such as thosecomprising of heavy (V_(H)) and light (V_(L)) chain variable domainsjoined by a linker or a complexed multimeric/multivalent constructsthereof, for example, diabodies (bivalent dimer), triabodies (trivalenttrimer), or tetrabodies (tetravalent tetramer). Multimeric antibodyfragments may also be multispecific, for example, a bispecific diabodymay comprise of two fragments each with specificity for a differentantigen. Further preferred antibody fragments include single domainantibodies (daBs) such as those comprising a single V_(H) or V_(L)domain capable of specifically binding to an antigen. Antibody fragmentsalso within the scope of the invention include scFv-C_(H) dimerconstructs i.e. a minibody.

According to a further embodiment, the composition comprises anantigen-binding polypeptide or protein with a molecular mass selectedfrom at least 10 KDa, at least 15 kDa, at least 35 kDa, at least 50 kDa,at least 70 kDa, or at least 90 kDa. Also preferred is anantigen-binding polypeptide or protein with a molecular mass of at least100 kDa, such as 100-150 kDa, or even higher than 150 kDa. Particularlypreferred are antigen-binding polypeptides or proteins with a molecularmass in the range of 70 kDa to 160 kDa.

In a further embodiment of the invention, the antigen-bindingpolypeptide or protein may be selected from a fusion protein comprisingan antibody fragment. An antibody fragment may be fused to anotherbioactive protein or polypeptide fragment, for example, a polypeptidetoxin, enzyme, cytokine, membrane protein, etc. Examples of a fusionprotein comprising an antibody fragment include etanercept andatacicept. Etanercept is a recombinant human protein with a molecularmass of 150 kDa, comprising the ligand binding portion of 75 kDa tumornecrosis factor receptor (TNFR) fused to the Fc portion of IgG1.

In another embodiment of the invention, the antigen-binding polypeptideor protein may be selected from an antibody-drug conjugate, wherein theantigen-binding polypeptide or protein is covalently linked, for examplevia a linker or chemically cross-linking to a small molecule drug or aradiolabelled component such as radionuclides. Examples of antibody-drugconjugates include gemtuzumab ozogamicin, brentuximab vedotin,⁹⁰Y-labelled ibritumomab tiuxetan, ¹³¹I-labelled tositumomab,^(99m)Tc-labelled arcitumomab. As used herein, the term antibody-drugconjugate may also refer to an antigen-binding polypeptide or proteinthat is substantially chemically modified, for example, by PEGylation(e.g. certolizumab pegol, a PEGylated Fab′ fragment of a humanized TNFinhibitor monoclonal antibody) or lipidation.

As understood herein, antigen-binding polypeptides and proteins may bechimeric, humanized or human. Chimeric monoclonal antibodies, forexample, refer to hybrid monoclonal antibodies comprising domains orregions of the heavy or light chains derived from antibody sequencesfrom more than one species, for example from murine and human antibodysequences. Humanized monoclonal antibodies refer to those that arepredominantly structurally derived from human antibody sequences,generally with a contribution of at least 85-95% human-derivedsequences, whereas the term human refer to those are derived solely fromhuman germline antibody sequences. In a preferred embodiment, thecompositions comprise of an antigen-binding polypeptide or proteinselected from a monoclonal antibody, wherein the monoclonal antibody isa chimeric, humanized, or human antibody.

In another embodiment, the composition may comprise a polyclonalantibody, or a heterogenous mixture of antibodies capable of recognizingmore than one epitope of an antigen.

In a preferred embodiment, the antigen-binding polypeptide or protein isa therapeutic or diagnostic compound or a vaccine. As used herein, atherapeutic compound is a compound that is useful for preventing adisease or condition, alleviating any symptoms of a disease orcondition, improving any disease or condition, delaying the progress ofa disease or condition or the like. A diagnostic compound is useful fordetermining the state of an organism, or for diagnosing a disease,condition, symptom, or patient phenotype. The therapeutic compound mustbe administered to the patient, whereas the diagnostic agent may be usedin vivo or in vitro, depending on the specific case. For the avoidanceof doubt, the therapeutic or diagnostic compound is incorporated withinthe composition of the invention in a therapeutically or diagnosticallyeffective amount.

In a particularly preferred embodiment, the compositions of theinvention comprise a monoclonal antibody or antibody fragment that istherapeutically effective or which may be administered for the treatmentof a disease or condition, such as an autoimmune disease or inflammatorycondition, a neurological disorder, or cancer. Exemplary monoclonalantibodies or antibody fragments for the treatment of cancer includealemtuzumab, bevacizumab, cetuximab, gemtuzumab, ipilimumab,ibritumomab, nimotuzumab, ofatumumab, panitumumab, rituximab,tositumomab, and trastuzumab. Exemplary monoclonal antibodies orantibody fragments for the treatment of autoimmune or inflammatoryconditions include adalimumab, alemtuzumab, belimumab, briakinumab,canakinumab, eculizumab, epratuzumab, efalizumab, golimumab, infliximab,mepolizumab, natalizumab, ofatumumab, ocrelizumab, otelixizumab,omalizumab, reslizumab, rituximab, teplizumab, tocilizumab, ustekinumab,and vedolizumab. Further examples of monoclonal antibodies or antibodyfragments which may be administered for the treatment, prevention ordiagnosis of a disease or condition include basiliximab, daclizumab,denosumab, eculizumab, palivizumab, and motavizumab.

Suspension or dispersion compositions according to the invention may inparticular comprise a cancer therapy agent selected from anantigenbinding polypeptide or protein such as a monoclonal antibody,polyclonal antibody, an antibody fragment, a fusion protein comprisingan antibody fragment, an antibody-drug conjugate, or any combinationthereof and a liquid vehicle comprising a semifluorinated alkane of theformula RFRH wherein RF is a linear perfluorinated hydrocarbon segmentwith 4 to 12 carbon atoms, and wherein RH is a linear alkyl group with 4to 8 carbon atoms. Antigen binding polypeptides or proteins which act asangiogenesis inhibitors or which are able to inhibit tumour cellproliferation (anti-proliferative agent) are particularly relevant. Theantibody fragment may be a fragment antigen-binding (Fab), asingle-chain variable fragment (scFv), a single-domain antibody, aminibody, or a diabody.

For example, a composition according to the invention may comprise ofbevacizumab and a liquid vehicle, wherein the liquid vehicle comprises asemifluorinated alkane of the formula RFRH, wherein RF is a linearperfluorinated hydrocarbon segment with 4 to 12 carbon atoms, andwherein RH is a linear alkyl group with 4 to 8 carbon atoms; and whereinbevacizumab is incorporated in the composition in the form a dispersionor suspension. Antigen-binding fragments of bevacizumab are alsocontemplated. Bevacizumab (tradename Avastin®) is a humanized murineantibody that targets VEGF-A (vascular endothelial growth factor A) andwhich acts as an angiogenesis inhibitor. Compositions of the inventioncomprising bevacizumab may be used for the treatment or prevention ofdiseases and conditions such as colorectal, lung, breast, renal or brain(glioblastoma) cancers, as well as eye conditions such as age-relatedmacular degeneration (AMD).

Further compositions may comprise the antibodies known under thepipeline name as Fsn0503 and Fsn1006. Fsn1006 is a dual-specificantibody that can bind to the human EGFR (epidermal growth factorreceptor) ligands amphiregulin and HB-EGF (heparin-binding epidermalgrowth factor), and which can act to inhibit cell-proliferation. Fsn1006is a humanized IgG1/kappa isotype. It has been demonstrated that Fsn1006works independently of the K-ras mutational status of the cell andtherefore has significant advantages over current EGFR targetingproperties such as cetuximab. Fsn0503 is also a humanized IgG1/kappaantibody that targets and inhibits the proteolytic activity of humanCathepsin S. Fsn0503 may be used in the treatment of cancer, and otherangiogenesis related diseases, in particular diseases where CathepsinS-mediated remodelling of the extracellular matrix is involved.

In a yet a further preferred embodiment, suspension or dispersioncompositions according to the invention may comprise a TNF inhibitorselected from an antigen-binding polypeptide or protein such as anmonoclonal antibody, polyclonal antibody, an antibody fragment, a fusionprotein comprising an antibody fragment, an antibody-drug conjugate, orany combination thereof, and a liquid vehicle comprising asemifluorinated alkane of the formula RFRH wherein RF is a linearperfluorinated hydrocarbon segment with 4 to 12 carbon atoms, andwherein RH is a linear alkyl group with 4 to 8 carbon atoms. ExemplaryTNF inhibitors are infliximab, etanercerpt and certolizumab and theirbiosimilars. Compositions of the invention may comprise these TNFinhibitors and a liquid vehicle, wherein the liquid vehicle consists ofsemifluorinated alkane selected from F4H5, F4H6, F4H8, F6H4, F6H6, F6H8,and F6H10. In particular, these compositions may be used in the therapyof autoimmune diseases affecting the gastrointestinal system such asCrohn's disease, ulcerative colitis, or conditions affecting the jointsand skin such as rheumatoid arthritis, psoriatic arthritis, ankylosingspondylitis and plaque psoriasis.

In yet another embodiment, the compositions of the invention comprise anantigen-binding polypeptide or protein at a concentration of preferablyat least 0.5 mg/mL, such as 0.5-10 mg/ml. In further preferredembodiments, the concentration is at least 1 mg/mL, at least 5 mg/mL, atleast 10 mg/mL, at least 15 mg/mL, at least 25 mg/mL or at least 35mg/mL.

In a further aspect, the current invention presents a method ofstabilizing an antigen-binding polypeptide or protein, comprising thestep of mixing the antigen-binding polypeptide or protein with a liquidvehicle comprising a semifluorinated alkane. The semifluorinated alkaneis of the formula RFRH, wherein RF is a linear perfluorinatedhydrocarbon segment with 4 to 12 carbon atoms, and wherein RH is alinear alkyl group with 4 to 8 carbon atoms. According to the method,the step of mixing the antigen-binding polypeptide or protein with theliquid vehicle is performed such as to form a suspension or adispersion. Such method of stabilizing an antigen-binding polypeptide orprotein may be useful for the preparation of a composition for use as amedicine, such as in the treatment, prevention or diagnosis of a diseaseor condition in a patient in need thereof. Optionally, such method ofstabilization may also be used for the preparation, manufacture orsynthesis of an antigen-binding polypeptide or protein.

As used herein, the term stability is defined as the maintenance of thechemical or physical integrity and/or bioactivity of the antigen-bindingpolypeptide or protein over a period of time. Stabilizing anantigen-binding polypeptide or protein includes the prevention or delayof degradation or deterioration of the antigen-binding polypeptide orprotein from its biologically and/or therapeutically active form to aninactive form. Instability may arise from events such as aggregation,denaturation, fragmentation, or chemical modifications such asoxidation, cross-linking, deamidation and reactions with othercomponents featured in the composition comprising the antigen-bindingpolypeptide or protein.

The stability of the antigen-binding protein or polypeptide in thecomposition may be characterized using known methods in the art,including but not limited to, measurement of biological activity such asantigen-binding activity with immunoassay techniques such as ELISA, orother techniques of determining purity or physical/chemical changes tothe antigen-binding protein or polypeptide such as size exclusionchromatography, capillary gel electrophoresis, circular dichroism, ormass spectrometry. Stability is determined by comparison of measurementsobtained via these types of characterization methods at an initial timepoint, such as at the time of formulation or preparation of thecomposition (i.e., as the case may be, the suspension or dispersion),and those obtained at a later time point, that is, after storage in agiven environment or condition.

It has been found by the inventors that an antigen-binding protein in aliquid vehicle comprising a semifluorinated alkane remains stable at 25°C. for at least 6 months. More remarkably, the antigen-binding proteinwas comparably stable when stored at a temperature of 40° C. over thesame period of time. That is, the composition comprising theantigen-binding protein retained effectively the same or similarantigen-binding activity to its initial antigen-binding activity.

In a preferred embodiment, the compositions of the invention retain atleast 85% or at least 90%, such as 90-95%, or even more than 95% oftheir initial antigen-binding activity during storage of 3 months at 25°C., or at room temperature, or at a temperature between room temperatureand 40° C. In another preferred embodiment, the compositions of theinvention retain at least 85% or at least 90%, such as 90-95%, or evenmore than 95% of their initial antigen-binding activity, during storageof 6 months at 25° C., or at room temperature (RT), or at a temperaturebetween RT and 40° C. In yet another embodiment, the compositions of theinvention retain at least 85% of their initial antigen-binding activity,during storage of 6 months between RT and 40° C. and a relative humidityof between 50-75%. In other embodiments, the time period of storage maybe 4-6 weeks, 6-12 weeks, or 3-6 months or 6-12 months. In furtherembodiments, the humidity (RH) during storage may be at least 40% or atleast 50%, or at least 65% or at least 75%.

As mentioned, the antigen-binding polypeptide or protein is incorporatedin the composition such as to form a dispersion or suspension. In otherwords, the antigen-binding polypeptide or protein is dispersed orsuspended in the liquid vehicle comprising a semifluorinated alkane.Whether a suspension is formed upon dispersing the antigen-bindingprotein in the liquid carrier depends e.g. on the nature of theantigen-binding protein, its concentration in the carrier, and theselected SFA(s).

As used herein, a suspension may be defined as a type of dispersion,i.e. a system having at least one continuous (or coherent) phase and atleast one discontinuous (or inner) phase which is dispersed in thecontinuous phase. In a suspension, the dispersed phase is in the solidstate. The suspensions useful for practising the invention are liquids,at least at physiological temperature, which means that the continuousphase is a liquid. Typically, the suspensions are also liquid at roomtemperature. Beyond suspensions, the term dispersions is understood toinclude colloidal systems in which an antigen-binding protein andpolypeptide is finely dispersed in the liquid phase. In someembodiments, the antigen-binding polypeptide or protein is also at leastpartially solvated.

A stabilized suspension or dispersion of an antigen-binding polypeptideor protein is prepared via a method comprising the step of mixing theantigen-binding polypeptide or protein with a liquid vehicle comprisinga semifluorinated alkane. The stability of the resulting suspension ordispersion may be characterized by the measurement of various physicalattributes including but not limited to, for example there-dispersibility of the suspended particles, particle size distributionand particle size growth over time, using such methods as those known inthe art.

In one particular embodiment, the composition comprises only theantigen-binding polypeptide or protein and one or more SFAs, i.e. thecomposition consists of the antigen-binding polypeptide or protein andone or more SFAs as defined above. In another preferred embodiment, thecomposition comprising an antigen-binding polypeptide or protein and oneor more SFAs is effectively or substantially free of water, i.e. thecomposition comprises no water, except perhaps for residual amounts ofwater introduced via other solid or liquid components or theantigen-binding polypeptide or protein itself. In other cases, thesuspension or dispersion compositions comprising an antigen-bindingpolypeptide or protein and a liquid vehicle comprising a semifluorinatedalkane may be water-free.

In contrast to some other suspensions or dispersions known in prior art,the formulations of the invention require no surfactant, or only smallamounts of surfactant, for their physical stabilisation. This is asignificant advantage as surfactants have a substantial potential forirritation and local toxicity, especially when administered bysubcutaneous or intramuscular injection or by instillation into the eye.According to one of the preferred embodiments, the compositions of theinvention are substantially free of surfactant. In a further embodiment,the total amount of surfactant or surfactants, if more than onesurfactant is incorporated, is not more than about 10 wt.-%, inparticular not more than about 5 wt.-%, or preferably not more thanabout 2 wt.-%, respectively. In further preferred embodiments, theamount is not more than about 1 wt.-%, or not more than about 0.5 wt.-%,respectively. In this context, the SFAs as described herein, althoughthey possess some amphiphilic properties due to their chemical structurewhich includes fluorinated and non-fluorinated alkyl (or alkylene)groups characterised by different degrees of lipophilicity, are notunderstood as being within the scope of surfactants.

The surfactants which are absent or only present in small amountsinclude non-ionic, cationic, anionic, and zwitterionic surfactants ascommonly used as excipients in various types of pharmaceuticalcompositions, e.g. as wetting agents, emulsifiers, dispersing agents,solubilisers and the like. Examples of surfactants which are consideredpotentially useful include tyloxapol, poloxamers such as Pluronic F68LFor Lutrol F68, Pluronic L-G2LF and Pluronic L62D, polysorbates such aspolysorbate 20 and polysorbate 80, polyoxyethylene castor oilderivatives, sorbitan esters, polyoxyl stearates, lecithins, purified orsynthetic phospholipids, and mixtures of two or more thereof.

The compositions of the invention may optionally comprise anon-fluorinated organic liquid, for example in order to modify theproperties of the liquid vehicle, such as the viscosity. Such otherliquid may be an oil selected from glyceride oils, liquid waxes, andliquid paraffin, or an organic solvent exhibiting a high degree ofbiocompatibility, or a mixture of more than one liquid excipients.

Examples of potentially useful oily excipients which may be used incombination with one or more SFA's include triglyceride oils (i.e.soybean oil, olive oil, sesame oil, cotton seed oil, castor oil, sweetalmond oil), mineral oil (i.e. petrolatum and liquid paraffin), mediumchain triglycerides (MCT), oily fatty acids, isopropyl myristate, oilyfatty alcohols, esters of sorbitol and fatty acids, oily sucrose esters,or any other oily substance which is physiologically tolerated by theeye.

Examples of potentially useful organic solvents include glycerol,propylene glycol, polyethylene glycol, and ethanol. The concentration ofthe cosolvent should preferably be low relative to that of the SFA orSFA mixture. If an organic solvent such as ethanol is used, it isrecommendable to keep it below a level of approx. 5 wt.-%. Morepreferably, the content of ethanol is from about 0.1 to about 2 wt.-%,and most preferably not more than about 1 wt.-%.

The composition may of course comprise further pharmaceutical excipientsas required or useful. Potentially useful excipients include acids,bases, antioxidants, stabilisers, synergists, colouring agents,thickening agents, and—if required in a particular case—a preservative.Generally, however, the invention provides a means of formulatingnon-aqueous compositions which are microbiologically stable. This is dueto the fact that SFAs are not normally prone to microbial contamination.Hence, it is possible to formulate preservative-free compositions to befilled in multi-use containers. Preservative-free compositions arebetter tolerated by many patients and enable lower costs of final goods.

The liquid suspensions of the invention may be prepared by conventionalmethods. In principle, the solid particles comprising the activeingredient may be dispersed in the liquid vehicle comprising the SFA.Alternatively, the particles may be precipitated in situ by addinga—typically organic—solution of the active ingredient (and, optionally,one or more solid excipients) under controlled conditions to theSFA-based vehicle.

The solid particles may be prepared by lyophilization or spray-drying ofa solution of the antigen-binding proteins or particles. The solutionmay be aqueous or non-aqueous and may further comprise pharmaceuticalexcipients as may be useful or required.

The particle size of the dispersed phase may be also adjusted before orafter the particles are combined with the liquid vehicle. In one of thepreferred embodiments, particles of the active ingredient are providedwhich already have the appropriately selected particle size. Powdershaving such selected particle size may be obtained directly from thesynthesis of the respective compound by crystal engineering, or aftersynthesis by conventional grinding or milling methods using standardequipment such as a ball mill, hammer mill, roller mill, colloidal mill,jet mill, or the like. If the particle size is to be reduced afterpreparation of a suspension, ultrasonication as well as various types ofhomogenisers may be used, such as colloid mills or high pressurehomogenisers.

The current invention also provides for a method for treating,preventing or diagnosing a disease or condition in a patient in needthereof, comprising the step of administering to the patient acomposition, preferably in the form of a suspension or dispersion,comprising an antigen-binding polypeptide or protein and a liquidvehicle, wherein the liquid vehicle comprises a semifluorinated alkane.The superior physical properties of the suspensions according to theinvention render these compositions particularly useful for topicaladministration to the eye of a patient, to the ear, nose or lung, orparenterally by injection. For example, the compositions of theinvention may be administered to the eye of a patient by topicalapplication or injection. Preferred modes of injection include dermal,subcutaneous, intramuscular, and locoregional injection. Most preferredare the subcutaneous and intramuscular routes of administration.

EXAMPLES Example 1

The stability of an anti-ECSCR (endothelial cell-specific chemotaxisregulator) monoclonal antibody (clone id 13G11 1A31 A7, a murine IgG/kantibody expressed from hybridoma cells that binds to the endothelialmarker ECSCR) in F6H8 at 25° C. and at 40° C. was studied over a periodof 6 months.

Lyophilized 0.25 mg samples of the anti-ECSCR monoclonal antibody(originally stored at −80° C. in PBS buffer) were reconstituted to aconcentration of 1 mg/mL in F6H8. The reconstituted samples were storedin crimped glass vials at 25° C./60% RH and at 40° C./75% RH. Thebinding activities of these samples were determined after 3 months and 6months of storage.

To serve as a first control, other samples of the lyophilized antibodywere stored in the lyophilized form at −80° C. These were reconstitutedin PBS only prior to analysis. A second control consisted of samples ofthe antibody in PBS which were never lyophilized, but kept underrefrigeration at 4° C.

The binding activities of the antibody samples to ECSCR antigen weredetermined by ELISA using the following protocol:

A Nunc MaxiSorp™ flat bottom Elisa plate was prepared by coating withECSCR antigen and an unrelated negative antigen. The antigens werediluted in a coating buffer at a concentration of 1 μg/mL and incubatedwith the plates overnight at 4° C. The plates were then blocked with 200μL of 3% blocking solution and incubated at RT on a shaker for 1-2hours, followed by three washes with PBS-Tween 20 solution and blotteddry. The antibody samples were diluted to 1 μg/mL in PBS. 100 ng or 10ng of each diluted sample was added to a well coated with the ECSCRantigen and a well coated with the negative control antigen. The platewas incubated at RT for 1 h on a shaker, then washed with PBS-Tween 20and blotted dry. The antibody aliquots were probed with goat-anti-mouseIgG-HRP secondary antibody conjugate (Biorad, Catalogue Nr. 170-6516) ata 1:5000 dilution in PBS. The plates were incubated for 1 h at RT on ashaker, followed by three washes with PBS-Tween 20 solution and blotteddry. The plate was incubated at 37° C. for 10 min with 100 μL of TMB (3,3′, 5, 5′-tetramethylbenzidine), followed by addition of 50 μL/well of1M HCl. Absorbance was measured at 450 nm with a spectrophotometer.

Comparison to the binding activities determined at the initial timepoint of reconstitution and the control samples show that the bindingactivity (determined from average of three samples) of the antibody inF6H8 stored at 25° C. or even at the higher temperature of 40° C. ismaintained after storage over a 6-month period (FIG. 1). The bindingactivity is comparable to that observed for lyophilized anti-ECSCRmonoclonal antibody samples stored at −80° C. (Control 1, FIG. 1) andfor a never-lyophilized sample stored at 4° C. in PBS (Control 2, FIG.1.)

Example 2

The stability of bevacizumab, Fsn1006 and Fsn0503 was studied at 25° C.,50° C., and 70° C. over a period of 4 weeks.

Lyophilization Protocol:

Stock solutions of each of the antibodies listed above were obtained;Fsn1006 and Fsn0503 as solutions in PBS, bevacizumab (tradenameAvastin®) in its commercial storage buffer. An equivalent of 80 mg ofbevacizumab was transferred to >7000 MWCO dialysis tubing and dialysedfor 72 hours against 3×2 L volumes of PBS. The antibody solutions werediluted to 0.5 mg/ml in PBS and 500 μl of each solution was aliquotedinto individual 5-ml amber glass pharmaceutical grade vials forlyophilisation. Lyophilisation was done over a 48 hour cycle.

Resuspension of the Lyophilized Antibodies:

The lyophilized antibodies were suspended in F4H5, F6H8, 50% vol F4H5 inF6H8, PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄,pH=7.4) at a concentration of 0.5 mg/mL with careful vortexing.Resuspension of the lyophilized antibodies in PBS forms solutions, whilesuspensions were formed with the semifluorinated alkanes. The resultingsuspensions and solutions were stored in amber glass vials at 25° C. and50° C. for 4 weeks (28 days) After day 23, the 50° C. samples weresubjected to 70° C. conditions.

The binding activities of the antibody samples were determined by ELISAtesting at t=0 (immediately after resuspension), t=2 weeks and t=4weeks. As a control, lyophilized antibodies samples which were neverre-suspended (stored at −80° C.) and solutions of the antibodies in PBSwhich were never lyophilized (stored at 4° C.) were used.

ELISA Protocol:

For the ELISA testing of each antibody, a Nunc Maxisorp 96 well platewas coated with the target antigen by adding 100 ng/well of theappropriate antigen in 100 μl 0.2 M carbonate buffer, pH 9.5 andincubated for 1 hour at 37° C. After washing with PBS containing 0.1%tween 20 (PBS-T), the plate was blocked by adding 200 μl 4% milk powderin PBS and incubating at room temperature for 2 hours on a shaker. Afterfurther washing, the antibody samples were applied. 1 ml of ×0.5 PBS wasadded to each non-aqueous vial. The antibody was extracted into thesolution by gentle rocking for 5 minutes. 1 μl of the aqueous layer wasthen transferred to a vial containing 999 μl PBS (to give a nominalvalue of 50 ng/well). 100 μl of each sample vial was then plated in 6replicates of 100 μl. The plates were incubated overnight at 4° C. Afterwashing, the secondary antibody was applied (goat anti-human HRPconjugate) at a 1:60,000 dilution in PBS; 100 μl was added to each welland the plate was incubated for 1 hour at room temperature with shaking.The plates were then washed with 3 volumes of PBS-T followed by 2volumes of PBS. 100 μl of TMB solution was then applied and the plateincubated for 10 minutes at 37° C. The reaction was stopped by theaddition of 50 μl 1M HCl. Absorbance at λ=450 nm was read for each well.

TABLE 1 Bevacizumab Activity (Mean OD ± 2SD at 450 nm) 50% vol. WeekF4H5 F6H8 F4H5 in F6H8 PBS 25° C. 0 3.15 ± 0.19 3.26 ± 0.11 3.12 ± 0.123.04 ± 0.03 2 3.23 ± 0.05 2.34 ± 0.09 3.05 ± 0.10 2.12 ± 0.07 4 2.16 ±0.36 2.23 ± 0.46 2.03 ± 0.56 0.30 ± 0.54 50° C. (70° C. last 5 days) 03.16 ± 0.01 3.26 ± 0.11 3.14 ± 0.05 3.00 ± 0.05 2 3.27 ± 0.09 3.19 ±0.10 3.06 ± 0.19 3.16 ± 0.08 4 1.63 ± 0.62 1.69 ± 0.34 1.64 ± 0.54  0.05± 0.003

TABLE 2 Fsn1006 Activity (Mean OD ± 2SD at 450 nm) 50% vol. Week F4H5F6H8 F4H5 in F6H8 PBS 25° C. 0 2.57 ± 0.20 2.47 ± 0.94 2.12 ± 0.21 2.63± 0.21 2 3.27 ± 0.13 3.32 ± 0.15 3.26 ± 0.10 2.99 ± 0.10 4 2.54 ± 0.302.43 ± 0.45 2.45 ± 0.38 0.62 ± 1.26 50° C. (70° C. last 5 days) 0 2.56 ±0.27 2.47 ± 0.94 2.26 ± 0.20 2.56 ± 0.24 2 3.32 ± 0.04 3.28 ± 0.04 2.81± 0.04 2.79 ± 0.13 4 2.01 ± 0.40 1.97 ± 0.24 1.80 ± 0.91 0.05 ± 0.01

TABLE 3 Fsn0503 Activity (Mean OD ± 2SD at 450 nm) 50% vol. Week F4H5F6H8 F4H5 in F6H8 PBS 25° C. 0 3.07 ± 0.10 3.06 ± 0.08 2.98 ± 0.22 3.11± 0.16 2 3.22 ± 0.23 3.22 ± 0.09 3.20 ± 0.08 2.01 ± 1.23 4 2.70 ± 0.192.43 ± 0.19 2.87 ± 0.24 0.72 ± 1.98 50° C. (70° C. last 5 days) 0 3.09 ±0.08 3.06 ± 0.08 3.07 ± 0.08 3.11 ± 0.07 2 3.26 ± 0.10 3.22 ± 0.12 3.18± 0.14 3.10 ± 0.21 4 2.64 ± 0.22 2.19 ± 0.18 2.73 ± 0.19 0.65 ± 1.91

The activity of bevacizumab, Fsn1006 and Fsn0503 stored as suspensionsin F4H5, F6H8 and 50% vol F4H5 in F6H8 was substantially consistentduring a 4-week period at both 25° C. and at 50° C., including theincrease to a storage temperature of 70° C. for the 50° C. sample duringthe last five days of the period. In contrast the binding activitydemonstrated by the solutions of these antibodies in PBS bufferdeteriorated significantly by the end of the four-week period regardlessof storage temperature.

Example 3

The binding activity of bevacizumab, Fsn1006, and Fsn0503 stored in SFAand PBS buffer at temperatures between 50° C. and 80° C. was examined.

The lyophilized antibodies bevacizumab, Fsn1006, and Fsn0503 (asprepared above in Example 2) were re-suspended in F4H5, F6H8, and PBS ata concentration of 0.5 mg/ml with careful vortexing. Re-suspension ofthese lyophilized antibodies in PBS yields solutions, while suspensionswere formed with the SFAs.

The suspension/solution samples were held at temperatures of 50° C., 55°C., 60° C., 65° C., 70° C., 75° C., and 80° C. respectively for a periodof 2 h and then cooled to 10° C. prior to PBS buffer extraction andactivity assay using the ELISA method as described above in Example 2.Each experiment was performed in triplicate. Samples heated at 40° C.were used as a control.

It was found that the bevacizumab (FIG. 2), Fsn1006 (FIG. 3) and Fsn0503(FIG. 4) suspensions in F4H5 and F6H8 demonstrated significant stabilitytowards thermal denaturation compared to the samples in aqueous buffer.Binding activity remained fairly constant between 50° C. and 80° C. forthe antibodies formulated in these semifluorinated alkanes. In contrast,the activity of these antibodies stored in aqueous PBS bufferdeteriorated sharply at temperatures above 60° C.

Example 4

The stability of a composition comprising an antigen-binding polypeptideor protein selected from infliximab (a chimeric monoclonal antibody withhuman constant and murine variable regions, and a molecular mass of 149kDa), the fusion protein etanercept (a recombinant human protein with amolecular mass of 150 kDa comprising the ligand binding portion of the75 kDa TNFR (tumor necrosis factor receptor) fused to Fc portion ofIgG1), and certolizumab (an antibody fragment with a molecular mass of91 kDa, comprising a recombinant Fab′ conjugated to an approximately 40kDa polyethylene glycol) or biosimilars thereof and a liquid vehiclecomprising a semifluorinated alkane of formula RFRH wherein RF is alinear perfluorinated hydrocarbon segment with 4 to 12 carbon atoms, andwherein RH is a linear alkyl group with 4 to 8 carbon (for example F4H5and F6H8) is studied and compared to formulations in different media.

The antibody is lyophilized and re-suspended in a semifluorinatedalkane. Comparative suspensions/solutions are also prepared in othermedia. The following stability tests are performed on theseformulations:

-   -   Storage stability under ICH conditions (25 and 40° C.).    -   Temperature stability outside of ICH conditions, i.e. thermal        denaturing

These are carried out in analogy to the methods described in examples1-3. Analytical methods suitable for assaying the stability of theseantigen-binding polypeptides are performed. Assays to monitor theactivity and potency of the antibody, as well as to monitor aggregationlevels throughout the course of the stability experiments listed aboveinclude techniques such as ELISA in similarity to the protocol asdescribed above in Examples 1-3.

The analytical methods are carried out with samples obtained directlyfrom the antibody suspensions or depending on the analytical technique,on samples which are aqueous buffer extractions.

It is expected that the compositions comprising the antigen bindingpolypeptide or proteins will exhibit improved stability and a decreasedpropensity towards aggregate formation.

1. Composition comprising an antigen-binding polypeptide or protein anda liquid vehicle, wherein the liquid vehicle comprises a semifluorinatedalkane of the formulaRFRH wherein RF is a linear perfluorinated hydrocarbon segment with 4 to12 carbon atoms, and wherein RH is a linear alkyl group with 4 to 8carbon atoms; and wherein the antigen-binding polypeptide or protein isincorporated in the composition such as to form a dispersion orsuspension.
 2. The method according to claim 10, wherein theantigen-binding polypeptide or protein is selected from a monoclonalantibody, polyclonal antibody, an antibody fragment, a fusion proteincomprising an antibody fragment, an antibody-drug conjugate, or anycombination thereof.
 3. The method according to claim 2, wherein theantibody fragment is a fragment antigen-binding (Fab), a single-chainvariable fragment (scFv), a single-domain antibody, a minibody, or adiabody.
 4. The method according to claim 2, wherein the monoclonalantibody is a chimeric, humanized, or human antibody.
 5. The methodaccording to claim 10, wherein the antigen-binding polypeptide orprotein has a molecular mass of at least 90 kDa.
 6. The method accordingto claim 10, wherein the semifluorinated alkane is selected from F4H5,F4H6, F4H8, F6H4, F6H6, F6H8, and F6H10.
 7. The method according toclaim 10, wherein the composition is substantially free of water.
 8. Themethod according to claim 10, wherein the composition retains at least85% of its initial antigen-binding activity during storage of 6 monthsat a temperature of between RT to 40° C.
 9. The method according toclaim 10, wherein the antigen-binding polypeptide or protein is at aconcentration of at least 0.5 mg/mL.
 10. A method for treating orpreventing a disease or condition in a patient in need thereof,comprising the step of administering to the patient an effective amountof a composition comprising an antigen-binding polypeptide or proteinand a liquid vehicle, wherein the liquid vehicle comprises asemifluorinated alkane of the formulaRFRH wherein RF is a linear perfluorinated hydrocarbon segment with 4 to12 carbon atoms, and wherein RH is a linear alkyl group with 4 to 8carbon atoms; and wherein the antigen-binding polypeptide or protein isincorporated in the composition such as to form a dispersion orsuspension.
 11. The method according to claim 10, wherein thecomposition is administered by subcutaneous, dermal, intramuscular, orlocoregional injection.
 12. A method of stabilizing an antigen-bindingpolypeptide or protein, comprising the step of mixing theantigen-binding polypeptide or protein with a liquid vehicle comprisinga semifluorinated alkane of the formulaRFRH wherein RF is a linear perfluorinated hydrocarbon segment with 4 to12 carbon atoms, and wherein RH is a linear alkyl group with 4 to 8carbon atoms, such as to form a suspension or a dispersion.
 13. Themethod of claim 12, wherein the antigen-binding polypeptide or proteinretains at least 85% of initial antigen-binding activity during storageof 6 months at a temperature between RT to 40° C.
 14. The methodaccording to claim 10, wherein the disease or condition is cancer or anautoimmune disease.
 15. The method according to claim 10, wherein theantigen-binding polypeptide or protein is an angiogenesis inhibitor, ananti-proliferative agent or a TNF inhibitor.
 16. The method according toclaim 12, wherein the semifluorinated alkane is selected from F4H5,F4H6, F4H8, F6H4, F6H6, F6H8, and F6H10.
 17. The method according toclaim 12, wherein the composition is substantially free of water. 18.The method according to claim 12, wherein the antigen-bindingpolypeptide or protein is a monoclonal antibody, a polyclonal antibody,an antibody fragment, a fusion protein comprising an antibody fragment,an antibody-drug conjugate, or any combination thereof.
 19. The methodof claim 18, wherein the semifluorinated alkane is selected from F4H5,F4H6, F4H8, F6H4, F6H6, F6H8, and F6H10.
 20. The method of claim 12,wherein the antigen-binding polypeptide or protein is at a concentrationof at least 0.5 mg/mL.